Gregory R. Ferrier

A Cellular Mechanism for the Generation of Ventricular Arrhythmias by Acetylstrophanthidin

Transmembrane activity was recorded from canine false tendons bathed with Tyrodes solution at 37°C. Stimulus patterns provided a 3-second pause after every ten beats. Acetylstrophanthidin was infused at concentrations up to 2×10−7 g/ml. One or two transient depolarizations (TDs) followed the last driven response of each series. The appearance of TDs was associated with depression of normal phase-4 depolarization. The peak of the earliest TD (TD-1) occurred at an interval approximately equal to the basic cycle length. The later TD (TD-2) occurred at about twice the basic cycle length. Coupling intervals were determined primarily by the last cycle length. The amplitude of TD-1 was maximal when the basic cycle length was 600 msec, but TD-2 continued to increase as the basic cycle length diminished further. The amplitude of both TDs increased with the number of beats in the train. Either or both could reach threshold and induce single extrasystoles or trains of extrasystoles. TDs could be induced to reach threshold after each driven response, resulting in sustained bigeminal rhythms with fixed coupling. Possibly TDs provide a mechanism for various clinically observed arrhythmias induced by cardiac glycosides.

Possible mechanisms of ventricular arrhythmias elicited by ischemia followed by reperfusion. Studies on isolated canine ventricular tissues.

The purpose of this study was to develop an isolated tissue model in which arrhythmic activity could be generated in response to conditions encountered in ischemia followed by reperfusion, and in which intracellular recordings could be used to identify and study arrhythmogenic mechanisms. Isolated canine Purkinje fiber-papillary muscle preparations were superfused with modified Tyrodes solutions. Tissues were exposed to conditions observed in ischemia (hypoxia, acddosis, elevated lactate, zero substrate for 40 minutes). Supervision with Tyrodes solution of ‘normal’ composition was then reinstituted. Transmembrane recordings from Purkinje and muscle tissues were made, using standard microelectrode techniques. Ischemic conditions caused loss of membrane potential, shortened action potentials, depressed excitability, and progressive bidirectional conduction block between muscle and Purkinje tissues. Spontaneous activity, probably reentrant in origin, was observed. Return to nonischemic conditions resulted in a multiphasic sequence of responses in Purkinje fibers: prompt hyperpolarization, progressive depolarization to unresponsiveness, and final repolarization to control. The depolarization phase was accompanied by oscillatory afterpotentials which initiated extrasystoles. Final repolarization included a phase of automaticity at low membrane potentials, during which Purkinje tissue functioned as a parasystolic focus. Elevation of potassium concentration to 10 mM during the ischemic period did not alter the sequence of electrophysiological events during ischemic conditions or upon reperfusion. This study demonstrates that ischemia followed by reperfusion elicits an orderly sequence of electrophysiological events which may constitute important mechanisms of arrhythmia in vivo.

Effect of calcium on acetylstrophanthidin-induced transient depolarizations in canine Purkinje tissue.

The role of calcium ions (Ca2+) in the generation of transient depolarizations (TDs) by acetylstrophanthidin was examined. Transmembrane activity was recorded from isolated canine false tendons exposed to acetylstrophanthidin; concentrations from 7.5 × 10−8 to 2 × 10−7 g/ml caused TDs coupled to driven action potentials and depressed slow diastolic depolarization. TDs could reach threshold and induce extrasystoles. Elevation of the Ca2+ concentration increased the amplitude of TDs induced by acetylstrophanthidin. High Ca2+ concentration (12.5 mM) caused TDs and depression of slow diastolic depolarization in the absence of acetylstrophanthidin. Elevation of potassium (K+) concentration depressed and reduction of K+ concentration potentiated TDs caused by either acetylstrophanthidin or high Ca2+ concentration. The production of TDs and the depression of slow diastolic depolarization by acetylstrophanthidin were reversed by reduction of the Ca2+ concentration or addition of manganese (2 mM) to the superfusing Tyrodes solution. The results suggest that TDs and arrhythmias produced by acetylstrophanthidin may be caused by a transient Ca2+ influx.

Contractions in guinea‐pig ventricular myocytes triggered by a calcium‐release mechanism separate from Na+ and L‐currents.

1. Unloaded cell shortening and membrane currents were examined in isolated guinea‐pig ventricular myocytes at 37 degrees C using video edge detection and single‐electrode voltage clamp. 2. Inward Na+ currents were eliminated by lidocaine, tetrodotoxin, replacement of extracellular Na+ with choline chloride or sucrose, or by voltage inactivation of Na+ channels. In the absence of Na+ current, the threshold for contraction was approximately ‐50 or ‐55 mV. 3. Verapamil (5 microM) and nifedipine (2 microM) failed to inhibit contractions at negative membrane potentials when positive conditioning pulses were used to maintain intracellular Ca2+ stores via Na(+)‐Ca2+ exchange. In contrast, 200 microM Ni2+ inhibited these contractions. 4. Contractions were abolished when the extracellular solution was nominally Ca2+ free. However, contractions were restored by as little as 50 microM extracellular Ca2+. 5. Ryanodine (30 nM) completely abolished contractions initiated by depolarizing steps from ‐65 to ‐40 mV, but had minimal effects on contractions initiated by depolarizing steps from ‐40 to +5 mV. Subtraction of contraction‐voltage relations determined in the presence of ryanodine from control relations revealed a ryanodine‐sensitive component of contraction. This component activated at ‐55 mV and reached a plateau near ‐25 mV. 6. The amplitudes of contractions initiated by depolarizing steps from ‐40 mV were directly proportional to the magnitude of Ca2+ current (ICa). In contrast, contractions initiated by steps from either ‐55 or ‐65 mV were not proportional to ICa. These contractions appeared at potentials negative to the threshold for L‐type Ca2+ current, increased to a plateau at more positive potentials and did not decrease at potentials at which ICa decreased. 7. Subtraction of the contraction‐voltage relationship determined from a membrane potential of ‐40 mV from that at ‐55 mV revealed a component of contraction with a negative activation threshold whose amplitude was not proportional to inward current. The shape of this relationship was virtually identical to that of the ryanodine‐sensitive component of contraction. 8. This study identifies a component of contraction associated with Ca2+ release from sarcoplasmic reticulum (SR) which can be separated from other mechanisms of contraction on the basis of membrane potential. Our observations suggest that this voltage‐dependent release mechanism is a true trigger mechanism which activates a portion of cardiac contraction which is attributable to SR Ca2+ release.

Contribution of variable entrance and exit block in protected foci to arrhythmogenesis in isolated ventricular tissues.

Automatic foci with membrane potentials in the range characterized by depolarizationinduced automaticity exhibit entrance block. The present study demonstrates a role of variable entrance and exit block in arrhythmogenesis. We studied canine interventricular septa with the right bundle branch exposed, isolated false tendons and isolated feline papillary muscle using standard microelectrode techniques. Foci of automaticity were produced either by focal application of electric current or by exposure of the preparations to Tyrodes solution containing 1.5–2.0 mM KCI. Foci induced by mild depolarization exhibited entrance block with exit conduction and were subject to electrotonic modulation. With greater depolarization, varying degrees of exit block developed. Various rhythms, including Wenckebach periodicity, resulted. Delayed emergence of electrotonically accelerated activity led to closely coupled extrasystoles resembling reentrant activity. Exit conduction in some preparations was facilitated by enhanced normal pacemaker activity (membrane potentials −70 mVor greater) in tissue peripheral to the focus. Also, when there were two sites of automaticity separated by an area of depressed conduction, intermodulation between the two automatic regions generated complex arryhthmias. Shifts in maximum diastolic potential also changed conduction and led to changes in arrhythmic patterns. In some experiments, focal automaticity was terminated by single stimuli. We conclude that complex and variable behavior of automatic foci may result in activity with characteristics previously attributed to other arrhythmic mechanisms.

Ionic mechanisms of transient inward current in the absence of Na(+)‐Ca2+ exchange in rabbit cardiac Purkinje fibres.

1. Membrane currents were measured with a two‐microelectrode technique in voltage clamped rabbit cardiac Purkinje fibres under conditions known to cause intracellular calcium overload and to eliminate or minimize Na(+)‐Ca2+ exchange. 2. Increasing [Ca2+]o from 2.5 to 5 mM or above and substituting external sodium with either sucrose, choline or Li+ induced an oscillatory transient inward current (TI) which peaked 200‐300 ms after repolarization from a previous depolarizing pulse. The TI quickly disappeared upon return to normal Tyrode solution. Both the rate and configuration of action potentials of Purkinje fibres also returned to control upon return to Tyrode solution after 30 min of high Ca2+ exposure, if the Ca2+ concentration was 30 mM or less. 3. The TI in Na(+)‐free solution was Ca2+ dependent. Either zero or low (2.5 mM) [Ca2+]o, or replacement of [Ca2+]o by BaCl prevented induction of the TI current upon repolarization from a previous depolarizing pulse. 4. In the presence of 30 mM‐CaCl2 and with choline chloride as the substitute for NaCl, TI had a distinct reversal potential (Erev) of ‐25 mV. The time‐to‐peak TI, either inward or outward, did not shift significantly with change in voltage. Both inward and outward TI were simultaneously abolished by exposure to 1 microM‐ryanodine, suggesting they were both activated by transient release of Ca2+ from the sarcoplasmic reticulum. The occurrence of TI in the absence of [Na+]o is not compatible with an electrogenic Na(+)‐Ca2+ exchange mechanism. The existence of a clear‐cut reversal potential suggests that an ionic channel may be responsible for the TI under these conditions. 5. Both the magnitude of peak TI and the Erev were affected by changes of CaCl2 concentration. (i) Under steady‐state conditions, peak inward TI was significantly increased when the [Ca2+]o was elevated from 5 to 15 mM. The peak TI in the outward direction was significantly increased when [Ca2+]o was elevated from 15 to 30 mM; however, the difference in peak inward TI at 15 and 30 mM [Ca2+]o was small. (ii) Clear‐cut reversals of TI were found at Ca2+ concentrations of 10 mM (Erev = ‐19.5 mV) or greater, and elevation of [Ca2+]o to 20, 30, 50 and 105 mM shifted the Erev to more negative potentials. (iii) In the presence of 5 mM [Ca2+]o the inward TI declined to zero at about ‐30 mV, and test voltages between ‐55 and +5 mV failed to reveal a distinct outward TI.(ABSTRACT TRUNCATED AT 400 WORDS)

Role of cAMP‐dependent protein kinase A in activation of a voltage‐sensitive release mechanism for cardiac contraction in guinea‐pig myocytes

1 Ionic currents and unloaded cell shortening were recorded from guinea‐pig ventricular myocytes with single electrode voltage clamp techniques and video edge detection at 37 °C. Patch pipettes (1–3 MΩ) were used to provide intracellular dialysis with pipette solutions. 2 Na+ currents were blocked with 200 μm lidocaine. Contractions initiated by the voltage‐sensitive release mechanism (VSRM) and Ca2+‐induced Ca2+ release (CICR) in response to L‐type Ca2+ current (ICa,L) were separated with voltage clamp protocols. 3 Without 8‐bromo cyclic adenosine 3′,5′‐monophosphate (8‐Br‐cAMP) in the pipette, small VSRM‐induced contractions occurred transiently in only 13 % of myocytes. In contrast, large ICa,L‐induced contractions were demonstrable in 100 % of cells. 4 Addition of 10 or 50 μm 8‐Br‐cAMP to the pipette increased the percentage of cells exhibiting VSRM contractions to 68 and 93 %, respectively. With 50 μm 8‐Br‐cAMP, contractions initiated by the VSRM and ICa,L were not significantly different in amplitude. 5 8‐Br‐cAMP‐supported VSRM contractions had characteristics of the VSRM shown previously in undialysed myocytes. Cd2+ (100 μm) blocked ICa,L and ICa,L contractions but not VSRM contractions. 8‐Br‐cAMP‐supported contractions exhibited steady‐state inactivation with parameters characteristic of the VSRM, as well as sigmoidal contraction‐voltage relations. 6 Without 8‐Br‐cAMP in the pipette, contraction‐voltage relations determined with steps from a post‐conditioning potential (Vpc) of either −40 or −65 mV were bell shaped, with a threshold near −35 mV. With 50 μm 8‐Br‐cAMP in the pipette, contraction‐voltage relations from a Vpc of −65 mV were sigmoidal and the threshold shifted to near −55 mV. Contraction‐voltage relations remained bell shaped in the presence of 8‐Br‐cAMP when the Vpc was −40 mV. 7 H‐89, which inhibits cAMP‐dependent protein kinase A (PKA), significantly reduced the amplitudes of VSRM contractions by approximately 84 % with 50 μm 8‐Br‐cAMP in the pipette. H‐89 also significantly reduced the amplitudes of peak ICa,L and ICa,L contractions, although to a lesser extent. 8 We conclude that intracellular dialysis with patch pipettes disrupts the adenylyl cyclase‐PKA phosphorylation cascade, and that the VSRM requires intracellular phosphorylation to be available for activation. Intracellular dialysis with solutions that do not maintain phosphorylation levels inhibits a major mechanism in cardiac excitation‐ contraction coupling.

Contribution of Na+-Ca2+ Exchange to Stimulation of Transient Inward Current by Isoproterenol in Rabbit Cardiac Purkinje Fibers

Cellular mechanisms underlying beta-adrenergic stimulation of the arrhythmogenic transient inward current (TI) were investigated by using a two-microelectrode voltage-clamp technique in rabbit cardiac Purkinje fibers. TI induced by elevating [Ca2+]o to 30 mmol/L and substituting [Na+]o with N-methyl-D-glucamine (NMG) chloride had a distinct reversal potential (EREV) of -25 mV, suggesting that Na(+)-Ca2+ exchange was not the charge carrier for TI. In the absence of [Na+]o, isoproterenol (ISO, 0.01 to 5.0 mumol/L) had no effect on either inward or outward TI or on the current-voltage relation of TI. However, ISO (0.1 mumol/L) significantly increased both inward and outward TIs without affecting the EREV of TI, if [Na+]o was present. Pretreatment with propranolol (0.2 mumol/L) or atenolol (0.2 mumol/L) abolished the stimulatory effects of ISO. Addition of propranolol (0.2 to 0.5 mumol/L) after the effects of ISO had developed caused only partial reversal of TI stimulation. This indicates persistence of stimulatory effects downstream from the initial agonist-receptor interaction. Forskolin (1 mumol/L), a direct adenylate cyclase activator, also strongly increased both inward and outward TI in the presence of [Na+]o. These effects also were abolished when [Na+]o was substituted by NMG. Inward and outward TIs enhanced by either ISO or forskolin were reversed by two putative Na(+)-Ca2+ exchange blockers, dodecylamine (20 mumol/L) and quinacrine (20 mumol/L). These results suggest that beta-adrenergic stimulation of TI is mediated by the Na(+)-Ca2+ exchange; stimulation likely involves phosphorylation of the exchanger or some factor that modulates exchanger activity.

Conduction Block Associated with Transient Depolarizations Induced by Acetylstrophanthidin in Isolated Canine Purkinje Fibers

Transient depolarizations (TDs) appeared in canine Purkinje tissue but not in cardiac muscle during exposure to 1–2 × 10−7 g/ml of acetylstrophanthidin. At low levels of toxicity, the TDs were associated with a decrease in threshold and an increase in automaticity. As intoxication progressed, the TDs coincided with transient periods of increased threshold and conduction block. Propagation from muscle to Purkinje tissue was blocked during, but not before or after, the crest of the TDs. As a result of the interaction between temporal and spatial (electrotonic) factors, various patterns of block, including unidirectional block, were observed. At moderate frequencies 2:1 block was demonstrated, but at faster rates 1:1 transmission occurred. At a later stage of toxicity, repetitive stimulation led to progressive depolarization in Purkinje tissue followed by a period of block during which slow repolarization eventually restored excitability and conductivity. In the whole heart, conduction block could permit reentry and could be responsible for the ultimate transition from ventricular tachycardia to ventricular fibrillation.

Differential effects of docosahexaenoic acid on contractions and L-type Ca2+ current in adult cardiac myocytes

UNLABELLED Beneficial effects of n-3 polyunsaturated fatty acids in Ca2+ overload have been attributed to blockade of L-type Ca2+ current (I(Ca-L)). However, cardiac contractions may be maintained despite block of I(Ca-L). OBJECTIVE This study investigates the cellular basis by which docosahexaenoic acid (DHA), a representative n-3 polyunsaturated fatty acid, inhibits I(Ca-L) while preserving contraction. METHODS Experiments were conducted in adult guinea pig ventricular myocytes with Na+ currents blocked. Contractions initiated by the voltage-sensitive release mechanism (VSRM) and calcium-induced calcium release (CICR) triggered by I(Ca-L), were activated separately with voltage clamp techniques. RESULTS DHA (10 microM) inhibited I(Ca-L) and CICR contractions but not VSRM contractions. CICR contractions exhibited a bell-shaped voltage-dependence. However, in the presence of DHA, only contractions with a sigmoidal voltage-dependence characteristic of the VSRM remained. These contractions exhibited inactivation properties characteristic of the VSRM. DHA abolished I(Ca-L) elicited by test steps from -40 mV. Block was voltage-dependent, as residual I(Ca-L) was elicited by steps from -70 mV. Cd2+ inhibited residual current, but not contractions initiated by the same activation steps. CONCLUSION Preservation of VSRM contractions during block of I(Ca-L), may explain the ability of n-3 polyunsaturated fatty acids to inhibit Ca2+ influx while preserving cardiac contractile function.